TR-03347; No of Pages 5
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ARTICLE IN PRESS
Thrombosis Research (2008) xx, xxx–xxx
intl.elsevierhealth.com/journals/thre
REGULAR ARTICLE
Increased procoagulant cell-derived microparticles
(C-MP) in splenectomized patients with ITP ☆
V. Fontana a , W. Jy a , E.R. Ahn a , P. Dudkiewicz a , L.L. Horstman a ,
R. Duncan b , Y.S. Ahn a,⁎
a
Wallace H Coulter Platelet Laboratory, Division of Hematology/Oncology, University of Miami, Miller School of
Medicine, 1600 NW 10th Ave, Room 7109A, Miami, FL 33136, United States
b
Department of Epidemiology and Public Health, University of Miami, Miller School of Medicine, 1120 NW 14th street
Suite 1063, Miami, FL 33136, United States
Received 22 August 2007; received in revised form 21 December 2007; accepted 27 December 2007
KEYWORDS
Splenectomy;
ITP;
Microparticles;
Red cell microparticles;
Cardiovascular disease
Abstract
Background: Splenectomy is frequently employed for therapeutic and diagnostic
purposes in various clinical disorders. However its long-term safety is not well
elucidated. Although risk of infection by encapsulated organisms is widely
recognized, less well-known are risks of thrombosis and cardiovascular disease.
Methods: We investigated levels of cell-derived microparticles (C-MP) in 23
splenectomized ITP (ITP-S) and 53 unsplenectomized ITP patients (ITP-nS). Assay of
C-MP derived from platelets (PMP), leukocytes (LMP), red cells (RMP) and endothelial
cells (EMP) were performed by flow cytometry. Coagulation parameters included PT,
aPTT and activities of FVIII, IX and XI. Results of all measures were compared between
the two groups, ITP-S vs ITP-nS.
Results: Levels of all C-MP were higher in ITP-S than ITP-nS but only RMP and LMP
reached statistical significance (p = 0.0035 and p b 0.0001, respectively). The aPTT
was significantly shorter in ITP-S (p = 0.029). Interestingly, correlation analysis
revealed that RMP, but not other C-MP, were associated with shortening of aPTT
(p = 0.024) as well as with increased activities of factors VIII (p = 0.023), IX (p = 0.021)
and XI (p = 0.0089).
☆
This work was supported by the Wallace H Coulter Foundation and the Mary Beth Weiss Research Fund.
⁎ Corresponding author. Wallace H Coulter Platelet Laboratory, Division of Hematology/Oncology, Department of Medicine, University
of Miami, Miller School of Medicine, 1600 NW 10th Ave, Room 7109A, Miami, FL 33136, United States. Tel.: +1 305 243 6606; fax: +1 305
243 4975.
E-mail address: [email protected] (Y.S. Ahn).
0049-3848/$ - see front matter © 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.thromres.2007.12.022
Please cite this article as: Fontana V, et al, Increased procoagulant cell-derived microparticles (C-MP) in splenectomized patients
with ITP, Thromb Res (2008), doi:10.1016/j.thromres.2007.12.022
ARTICLE IN PRESS
2
V. Fontana et al.
Conclusions: RMP and LMP were significantly elevated in splenectomized compared to
non-splenectomized ITP patients. This suggests that the spleen functions to clear
procoagulant C-MP, and that elevation of C-MP might contribute to increased risk of
thrombosis, progression of atherosclerosis and cardiovascular disease following
splenectomy.
© 2008 Elsevier Ltd. All rights reserved.
Introduction
Splenectomy is widely employed for treatment of
various clinical disorders such as immune thrombocytopenic purpura (ITP), autoimmune hemolytic
anemia, hairy cell leukemia and other lympho- and
myelo-proliferative disorders. It is often beneficial
in improving cytopenias associated with hypersplenism. Splenectomy is also infrequently used for
diagnostic purposes such as staging of Hodgkin's
disease and splenomegaly of unknown causes [1].
Although some complications of splenectomy are
known, its overall long-term complications and
safety have not been well elucidated. Increased
susceptibility to infections of encapsulated microorganisms is a well-known complication, especially
in young patients, and recommendations of vaccination prior to surgery to prevent life-threatening
infections are widely publicized and practiced.
However, other potential complications have not
been systematically investigated. One epidemiologic study documented increased incidence of early
cardiovascular death among splenectomized individuals [2].
ITP is an autoimmune disease in which antiplatelet antibodies interact with platelets and the
opsonized platelets are recognized and removed
by macrophages, especially in the spleen, leading
to thrombocytopenia and a hemorrhagic diathesis
[3–5]. Splenectomy is widely employed as the
definitive measure for treatment of refractory ITP
[3,4]. Long-term complications of splenectomy in
ITP patients have not been studied systematically.
However, recent studies indicate that post-operative thrombotic complications are more prevalent
than generally appreciated [6–10].
Cell-derived microparticles (C-MP) are microvesicles of size b 1.0 µm, released from parent cells
during cell activation or apoptosis. Most C-MP are
highly procoagulant, expressing annexin V binding
sites and tissue factor, and are capable of interacting
with other cells to participate in various physiologic
and pathologic processes, especially thrombosis,
inflammation [11,12], and angiogenesis [13].
Recently, C-MP are receiving increasing attention
as sensitive biomarkers of prothrombotic and
inflammatory states [11,12,14,15]. However, rela-
tively little is yet known of their specific functions,
although new data supporting their likely roles in
thrombosis, inflammation and progression of atherosclerosis are accumulating in recent literature.
In the present study, we compare levels of C-MP
species from four cell types, and coagulation factor activities, in splenectomized (ITP-S) vs. nonsplenectomized (ITP-nS) ITP patients. We report
elevated levels of C-MP and enhanced blood
procoagulant activity in ITP-S compared to ITP-nS,
extending our preliminary report [16].
Materials and methods
Patient population
Seventy-six patients meeting the diagnostic criteria of ITP [17]
were recruited consecutively at the clinics and hospitals of
University of Miami's Miller School of Medicine. Twenty-three
patients had splenectomy (ITP-S) and 53 had not (ITP-nS). The
study was approved by the institutional review board and
informed consents were obtained.
The ITP-S patients included 7 males and 16 females, with
mean age of 55.6 yr. The ITP-nS group consisted of 21 males and
32 females, with mean age 56.7 yr. In the ITP-S group, 18 had
active ITP with persistent subnormal platelet count and 5 were in
remission. In the ITP-nS group, 33 had active ITP and 20 were in
remission. The duration of ITP since diagnosis ranged from 1 to
50 yr, with mean of 18.1 yr in the ITP-S group; and from 0.5 to
32 yr, with mean of 5.6 yr, in the ITP-nS group. When demographic
data on the two groups were compared, there was no significant
difference in age, sex ratio, or platelet counts between the
groups. However, the duration of ITP was significantly longer in
the ITP-S than in the ITP-nS group.
Laboratory studies
CBC and platelet counts, blood chemistry and blood coagulation
tests (PT, aPTT and activities of FVIII, FIX, FXI) were performed in
the clinical coagulation laboratory, Department of Pathology,
University of Miami Miller School of Medicine. Platelet counts
were confirmed by examination of blood smears. For blood
clotting tests, Platelin L (Biomerieux) was used as reagent for
aPTT and Simplastin HTF (Trinity Biotech) for PT. For functional
assay of factors VIII, IX and XI, deficient plasma were used with
PTT Automate (Diagnostica Stago).
Assay of C-MP
Blood was collected in citrate Vacutainers with application of
light tourniquet using a 21 gauge needle and the first tube was
discarded to avoid artifacts due to venipuncture. Within 3 h of
collection, the blood was centrifuged at 160 ×g for 10 min to
Please cite this article as: Fontana V, et al, Increased procoagulant cell-derived microparticles (C-MP) in splenectomized patients
with ITP, Thromb Res (2008), doi:10.1016/j.thromres.2007.12.022
ARTICLE IN PRESS
Splenectomy and cell derived microparticles
prepare platelet-rich plasma (PRP) and the PRP was further
centrifuged for 6 min at 2500 ×g to obtain platelet-poor plasma
(PPP). This speed preserved the majority of C-MP; residual
contaminating platelets were gated out in flow cytometry.
The flow cytometric assay of PMP and EMP briefly described
below was previously detailed and illustrated by representative
printouts [18]. Assay of RMP and LMP is essentially the same
except for the fluorescent antibodies used: FITC-labeled antiCD45 mAb for LMP and FITC anti-glycophorin mAb for RMP.
Fluorescence flow cytometry was used to assay C-MP. To
measure PMP and EMP simultaneously, 25 μL of PPP was
incubated with 4 μL of anti-human CD42-FITC and 4 μL of antiCD31-PE. Samples were incubated at room temperature for
20 min with gentle shaking, then 0.5 mL PBS was added and the
samples were ready for flow cytometry in a Coulter EPICS XL.
Detection of particles was by triggering on CD31-PE fluorescence
signal greater than that of matched isotype control from the
same supplier. All particles positive for CD31 were displayed in a
2-D scatter plot showing forward scattering (y-axis) vs. CD31
fluorescence (x-axis). Standard beads of 1.5 μm were used to set
the gating of MP b1.5 μm. The MP population with CD31+ and size
b1.5 μm were selected and displayed in a new scatter plot
showing CD42-FITC fluorescence (y-axis) vs. CD31-PE fluorescence
(x-axis). The CD31+/CD42+ and CD31+/CD42− MP were identified
as PMP and EMP, respectively. For measuring LMP or RMP, 4 μL of
anti-human CD45-FITC (LMP) or anti-human glycophorin (RMP) mAb
were incubated with 25 μL of PPP, then handled as described above.
All particles positive for CD45-FITC or glycophorin-FITC were
displayed in a 2-D scatter plot showing forward scatter (y-axis) vs.
FITC fluorescence (y-axis). Any particles b1.5 μm were counted as
LMP (CD45+) or RMP (glycophorin+).
Statistics
All continuous variables except age were non-normally distributed within one or both groups and transformations were not able
to achieve joint normality across groups. Consequently, major
comparisons were made using the Wilcoxon Rank Sums Test and
correlations were computed using the Spearman Rank Correlation. Multivariate tests relating the various C-MP fractions and
the clotting factors to the presence or absence of splenectomy
while adjusting for age, gender, remission status, and duration of
illness were performed using stepwise selection in the Multiple
Regression Model. Statistical significance required a p-value of
0.05 or less.
Results
The two groups of patients (ITP-S and ITP-nS) were
comparable in all parameters including age, sex, and
platelet counts, with the exception of duration of ITP,
which was significantly longer in ITP-S vs ITP-nS
(p b 0.0001). When we compared C-MP and coagulation
parameters in relation to duration of ITP, we found that CMP and clotting factors were not influenced by the
duration of ITP.
Table 1 summarizes data of C-MP and clotting
parameters between ITP-S and ITP-nS. The mean values
of all C-MP were higher in ITP-S compared to ITP-nS.
However, only RMP and LMP yielded statistical discrimination between the ITP-S vs. ITP-nS groups (p = 0.0035 and
p b 0.0001, respectively). Among the coagulation tests,
PT was similar between the groups but aPTT was
significantly shortened in ITP-S compared to ITP-nS
3
Table 1 Data on microparticles (RMP = red cell
microparticles, LMP = leucocytes microparticles,
EMP = endothelial microparticles, PMP = platelets
microparticles) and coagulation parameters in ITP
patients with splenectomy (ITP-S) vs. those with
no splenectomy (ITP-nS) are summarized in the
table
ITP-s (23)
ITP-ns (53)
P value
Mean ± standard Mean ± standard
error
error
Microparticles:
RMP
2552 ± 254
(U/µL)
LMP
1519 ± 83
(U/µL)
EMP
552 ± 131
(U/µL)
PMP
11390 ± 2801
(U/µL)
Coagulation
aPTT (s)
FVIII
(IU/mL)
FIX
(IU/mL)
FXI
(IU/mL)
1686 ± 114
0.0035
1184 ± 26
b 0.0001
379 ± 41
n.s.
11080 ± 1021
n.s.
measures:
23.9 ± 0.9
1.82 ± 0.2
25.6 ± 0.5
1.57 ± 0.1
0.029
n.s.
1.61 ± 0.1
1.39 ± 0.1
n.s.
1.25 ± 0.1
1.13 ± 0.1
n.s.
(p = 0.029). Activities of clotting factors VIII, IX, XI were
also higher in ITP-S compared to ITP-nS but did not reach
statistical significance.
Multivariate analysis revealed that the univariate
relations between C-MP and splenectomy or between
clotting factor activities and splenectomy did not reach
significance when adjusted for age, gender, remission
status, or duration of disease.
However, as shown in Fig. 1a, levels of RMP correlated
inversely with aPTT (p = 0.024), and in Fig. 1b, correlated
directly with FVIII (p = 0.023). Fig. 1c and d shows that RMP
levels correlated also with FIX (p = 0.021) and with FXI
(p = 0.0089). Interestingly, none of the other C-MP (i.e.,
PMP, LMP, EMP) correlated significantly with any of the
clotting parameters or shortening of aPTT.
Discussion
C-MP are procoagulant by expression of anionic
phospholipids such as phosphatidylserine (PS), on
which the coagulation proteins assemble, and by
transport of tissue factor (TF). The spleen is the
main organ for removal of RBC and other cells
expressing PS or opsonized with antibodies. Exposed
PS on cells is a signal for their clearance by
macrophages [19,20], therefore the spleen is likely
involved also in clearance of C-MP, consistent with
findings in the present study.
We reported in 1992 that PMP appear to be
hemostatically functional in patients with ITP [21]
Please cite this article as: Fontana V, et al, Increased procoagulant cell-derived microparticles (C-MP) in splenectomized patients
with ITP, Thromb Res (2008), doi:10.1016/j.thromres.2007.12.022
ARTICLE IN PRESS
4
V. Fontana et al.
Figure 1 Correlation analysis between RMP and aPTT and
clotting factor activities is shown. The analysis reveals that
RMP correlate inversely with aPTT (p = 0.024) in (a) and
directly with factors VIII (p = 0.023) in (b), IX (p = 0.021) in (c),
and XI (p = 0.0089) in (d). Other C-MP such as PMP, LMP, EMP
did not show significant correlation with any parameters (not
shown).
since those with high levels rarely bled, and those
with the highest levels often suffered TIA-like
syndromes due to ischemic small vessel disease
[21,22]. PMP can bind and activate neutrophils [23]
suggesting a role in inflammation as well [23,24],
and participate in angiogenesis [14].
Recently EMP have emerged as useful biomarkers of endothelial injury [12]. They exist in
multiple phenotypes, such as those arising from
activation vs. apoptosis [25], and have provided
insights on endothelial status in several vascular
disorders [12,15]. Some EMP transport vWF [26]
and can modulate coagulation and inflammation
[12,15].
LMP, such as from monocytes and neutrophils,
carry TF and are thought to be the main source of
circulating TF and to contribute to the progression
of atherosclerosis [27,28].
Of all C-MP types, RMP have received the least
attention. Cappellini et al., investigating hypercoagulability following splenectomy in patients with
thalassemia, indicated that release of procoagulant
phospholipids such as PS (platelet factor 3, PF3)
during hemolysis contributed to a hypercoagulable
state (HCS) post-splenectomy [29]. When RBC were
used as a source of phospholipids instead of
platelets in a prothrombinase assay, thrombin
generation was two-fold higher in splenectomized
than non-splenectomized patients [29]. In that
study, fibrinopeptide A, F1 + 2 and D-dimer were
significantly higher in the splenectomized patients
[29]. Similarly, Opartkiattikul et al. found elevated
PF3 activity in splenectomized vs. non-splenectomized patients with beta-thalassemia/HbE [30]. In
another study, levels of thrombin–antithrombin III
complex, prothrombin fragments, and expression of
PS on RBC were increased in patients with splenectomy [31].
An unexpected finding of the present study is
that of all C-MP assayed, only RMP were closely
associated with a trend of elevated clotting factor
activities and shortened aPTT. Thus, RMP may
contribute to HCS following splenectomy for ITP.
More recently, shortened aPTT predicted recurrence of venous thromboembolism [32], and elevated clotting factor activities were independent
risk factors for arterial and venous thrombosis
[33,34].
It was shown that C-MP from patients with
myocardial infarction caused endothelial dysfunction [35], and that exposure of rat aortic ring to EMP
inhibited NO synthesis and impaired acetylcholineinduced vaso-relaxation [36]. A role of EMP in
atherosclerosis and progression of vascular disorders has been proposed [36].
Thrombosis following splenectomy has been
reported in the literature of hematology [6,7]
and surgery [8–10]. In a prospective study, 60
consecutive patients with hematologic disorders
were investigated by serial doppler pre- and postsplenectomy, revealing higher incidence (6.7%) of
post-splenectomy portal and splenic vein thrombosis [10]. Thrombocytosis and elevation of C-MP
including RMP likely contribute to this heightened
risk.
Data is scanty on long-term effects of splenectomy
on atherosclerosis, although an epidemiologic study
of veterans who underwent splenectomy by war
trauma in 1932–1945 revealed a higher incidence of
cardiovascular death compared to non-splenectomized [2].
The present study provides new bases of increased risk of thrombosis and atherosclerosis
following splenectomy. However, this is a retrospective analysis in a limited number of patients.
Therefore, a large-scale prospective study is required to substantiate our data and the contribution
of C-MP in thrombogenesis and progression of
atherosclerosis in splenectomized patients.
Acknowledgments
This work was supported by the Wallace H Coulter
Foundation and the Mary Beth Research Fund.
Please cite this article as: Fontana V, et al, Increased procoagulant cell-derived microparticles (C-MP) in splenectomized patients
with ITP, Thromb Res (2008), doi:10.1016/j.thromres.2007.12.022
ARTICLE IN PRESS
Splenectomy and cell derived microparticles
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Please cite this article as: Fontana V, et al, Increased procoagulant cell-derived microparticles (C-MP) in splenectomized patients
with ITP, Thromb Res (2008), doi:10.1016/j.thromres.2007.12.022